This FastTrack Phase 1/2 application aims to create a new IND-ready CAR-T platform for the treatment of HIV, developed in the laboratory and tested in non-human primates. Molecular engineering techniques have been applied to create chimeric antigen receptors (CAR) expressed in T cells to target HIV-infected cells. CD4-based CARs are designed to achieve immune eradication of HIV1 infections through recognizing gp120 envelope protein on infected cells. However, prior clinical trials did not meet with success, which we propose to address with the plan of this research. One of the features predicted to affect efficiency of CD4 CAR is T cell exhaustion, characterized by high PD1 expression of HIV-specific CD8 and CD4 T cells. Our laboratory recently defined transcription factor YY1 to be master regulator of T cell exhaustion, mediating upregulation of checkpoint receptors (CR) and downregulation of Type I cytokines with accompanying cytotoxic failure. We confirmed that YY1 is increased in parallel with PD1 in CD4 T cells in chronic HIV infection. Knockdown of YY1 restored cytokine IL2 production in preclinical testing and reduced CR expression, including exhaustion marker PD1, where blocked PD1 and restored IL2 correlated with recovered T cell cytotoxic potency. Another drawback of prior CD4- based CAR-T is that CD8 T cells expressing the CD4 CAR receptor are now readily infected and eliminated by HIV that could also have hampered success of prior human trials. Lastly, prior tests involved 1st generation (gen) CARs of limited signaling potential that are now improved with addition of costimulation that may be yet further improved. Our overall goal is to create a more effective CAR-T for the control of HIV. Our Aims for this proposal include (1) creating anti-HIV CAR-T cells that will resist T cell exhaustion with incorporation of YY1 shRNA for sustained anti- HIV potency. Further, we will (2) render the CD4 CAR-T infection-proof with RNAi intervention to block infection and virus replication in the CAR-T. Finally, we will (3) conduct a complementary effort to generate new 3rd gen 3-signal CD4 CARs (CAR3) that incorporate additional costimulatory molecules to improve potency and reactivation potential. Building on our considerable preliminary work, we will quickly finish the Phase 1 component to complete molecular engineering efforts in months 1-6, moving directly to Phase 2 in vitro and then non- human primate testing. This plan is a collaboration between IT Bio, LLC (Boston) and the lab of Dr Steven Braun (Tulane). The Tulane National Primate Research Center (TNPRC) will conduct the NHP experiments under the direction of IT Bio, LLC through a fee-for-service contract. With these three efforts ? suppressing T cell exhaustion, rendering CAR-T infection-proof and increasing T cell potency and reactivation potential ? we hope to obtain a novel, IND-ready cellular therapeutic agent that will provide a sustained control of HIV to parallel recent successes in CAR- T treatment of B cell cancers.